High-resolution functional profiling of hepatitis C virus genome.
Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3' non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.
Arumugaswami
Vaithilingaraja
V
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America.
Remenyi
Roland
R
Kanagavel
Vidhya
V
Sue
Eric Yiang
EY
Ngoc Ho
Tuyet
T
Liu
Chang
C
Fontanes
Vanessa
V
Dasgupta
Asim
A
Sun
Ren
R
D024506:5' Flanking Region; D000465:Algorithms; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D020869:Gene Expression Profiling / Q000379:methods; D016679:Genome, Viral; D015698:Genomic Library; D023281:Genomics / Q000379:methods; D016174:Hepacivirus / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000235:genetics
D014764:Viral Proteins